The long-term objective of this project is the determination of the sequence of molecular events during duplication of Escherichia coli, and the biochemical mechanisms which control the initiation and completion of each of the major events in the sequence. A major objective for the current project is the determination of the molecular mechanisms which control the initiation of DNA replication. The mechanism which controls the timing of initiation of chromosome replication will be analyzed by performing temperature shifts in synchronous cultures of E. coli B/r containing dnaA or dnaC mutations. Previous work indicated that preincubation of a dnaC mutant at non-permissive temperature for 80 min. leads to at least two rounds of initiation of chromosome replication upon shift to permissive temperature in the absence of protein synthesis. The synthetic processes required for the second initiation event will be determined under conditions in which protein and DNA synthesis are totally inhibited. Another major aspect of this work concerns analysis of the second division, which takes place upon shift of dnaC mutants to non-permissive temperature. Of particular interest is whether this second division causes, or is a result of, initiation of a new round of chromosome replication. The timing of replication of F'lac plasmids during the division cycle of E. coli B/r will be determined by analysis of incorporation of radioactive thymidine into covalently closed circular DNA during synchronous growth. The number of chromosomes per cell, and the time for their replication, will be analyzed in three substrains of E. coli B/r growing at various rates by measurement of total DNA content in exponentially growing cultures.